This invention relates to methods of isolating a mutant cell containing one or more mutations which enhance production, excretion or secretion, of a desired compound from the cell compared to a cell without such mutations.
It is common to isolate mutant cells which overproduce a specific metabolite by selecting cells which are resistant to analogs of the metabolite. For example, Yamada et al. (Agric. Biol. Chem. (1983) 47:1011). describe the isolation of mutants which overproduce biotin by selecting for cells resistant to a biotin analog. Yamada et al. (Agric. Biol. Chem. (1982) 46:47), and Chattopadhyay, et al. (J. Gen. Microbiol. (1991) 137:685), isolated methionine-overproducing mutants by selecting cells resistant to ethionine. Hibberd (Iowa State J. Res. (1988) 62:479), isolated valine-overproducing mutants, selecting valine resistance). Kempe et al. (Cell (1976) 9:541), isolated pyrimidine nucleotide biosynthetic enzyme overproducers, selecting resistance to N-phosphonoacetyl-1-aspartate). Hall et al. (J. Bacter. (1989) 143:981), isolated amino acid overproducers in cyanobacteria by selecting antimetabolite analog resistance. In Hall et al. (id.) wild type Bacillus subtilis cells were used as a test lawn for screening obvious regulatory mutants from among collections of analog resistant strains. Auxotrophic strains of B. subtilis were convenient indicator strains for identification of mutants in Cyanobacteria through observation of syntrophic growth responses. Green (2d Chem. Congress N. Am. Continent, San Francisco, Aug. 22, 1989, Abst. No. 16) describes production of mutant corn cells resistant to lysine- and threonine-induced growth inhibition. Grull et al. (J. Bacter. (1979) 137:480,) describe isolation of amino acid overproducing mutants of E. coli obtained by mutagenesis and penicillin enrichment. Vincenzotto et al. 90 (Arch. Internat. de Chemi (1982) 90:B88) describe isolation of mutants of an alga by mutagenesis and screening on agar medium containing various dyes. Santhaguru et al. (Israel J. Med. Sci. (1985) 21:185,) describe use of levulinate, a competitive inhibitor of the heme biosynthetic pathway, for isolation of heme overproducing Rhizobium mutants.
Isolation of cells that make a desired product from a population of cells that do not make the desired product is a problem when the production of the desired compound does not endow the cell with any selective advantage. In these instances, one must develop a method of screening for production of the desired product. A screening method which employs a detector strain present in an overlay was utilized by Pai (J. Bacteriol. (1972) 112:1280) to isolate a strain that overproduces biotin. In this method, wild type E. coli were mutagenized and plated with an E. coli biotin auxotroph. The mutagenized E. coli was not itself an auxotroph, and thus, a mutual two-way symbiotic relationship was not achieved. A similar method has more recently been employed to obtain lysine excretors (LiMuti et al. (1989) Microbiol. Meth. 9:129). A variation of the overlay method has been developed where a micropore membrane is used to separate the strain from the substrate (U.S. Pat. No. 4,421,849). A problem with these strategies is the limitation on the number of cells that can be screened at one time. The limit is based on the need to distinguish individual colonies on a plate which places the limit, e.g. for bacteria, at 1,000 to 10,000 per each 100 mm diameter plate. If the event resulting in production of the desired compound occurs at a frequency of 10.sup.-7, then 1,000 plates would have to be screened to obtain one event.